A lipase mutant and its use for the biocatalytic production of dihydrohuperzine

Abstract

The application discloses a lipase mutant and a method for biocatalytically preparing dihydrohuperzine by using the same, and relates to the technical field of bioengineering. The application provides a lipase mutant, and the amino acid sequence of the lipase mutant is shown as SEQ ID NO:2. The application also provides a method for synthesizing dihydrohuperzine D by using the lipase mutant, realizes biosynthesis of dihydrohuperzine D, adopts one-step synthesis by using an enzyme, does not need a group protection and deprotection step, is green and environment-friendly in the production process, greatly reduces industrial wastewater discharge, and has important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a lipase mutant and its use in the preparation of dihydro avenanthramide by biocatalysis. BACKGROUND

[0002] Dihydro avenanthramide D is a nitrogen-containing phenolic acid derivative designed according to natural avenanthramide, has rapid anti-inflammatory and antipruritic activity, and is an important active ingredient of many anti-allergic skin care products. Its chemical structural formula is as follows:

[0003]

[0004] Dihydro avenanthramide D is an organic compound formed by connecting p-hydroxycinnamic acid and o-aminobenzoic acid with an amide bond (-HNCO-). It has strong antioxidant, lipid-lowering, anti-irritation, and antipruritic and anti-inflammatory effects, and therefore has very wide applications in the fields of beauty, care, and medicine. Since dihydro avenanthramide D has a low concentration in oats, its purification on an industrial scale requires high costs. Therefore, the current research focus on this type of substance is to develop a synthesis process that can be applied to industrial production. At present, the main method for synthesizing dihydro avenanthramide D is chemical synthesis. In the research report of Chinese patent CN 104418764B, p-hydroxybenzoic acid and o-aminobenzoic acid are used as raw materials, N,N carbonyl diimidazole is used as a condensing agent, and the condensation reaction is carried out in a pyridine reaction medium. However, this method has the disadvantages of low yield, large amount of wastewater pollution, and difficult separation of the product. In view of the above problems, Chinese patent CN 112939803B uses p-hydroxybenzoic acid and acetic anhydride as raw materials to prepare dihydro avenanthramide D in a three-step process in a chlorinated solvent. This process effectively reduces wastewater discharge and environmental pollution, improves product yield, and reduces production costs. However, this method has the disadvantages of complicated synthesis steps, dependence on phenolic hydroxyl protection process, and the need for strict control of reaction conditions at each step, which is not conducive to large-scale industrial synthesis.

[0005] Compared with traditional chemical synthesis, the high efficiency, high stereoselectivity and specificity of enzymes can effectively solve the above problems in the synthesis of dihydroheteroclausine D, so the biological method is a more ideal industrial alternative method. It is worth noting that in the enzyme-catalyzed synthesis system, the enzyme is limited by many factors in the aqueous reaction medium, including the difficulty of many compounds to dissolve in water, the occurrence of side reactions in water, the degradation of products and the difficulty of subsequent product purification in water. The above problems greatly limit the further industrial application of enzymes. In order to improve the enzyme catalytic process, many researchers prefer to use organic solvents as the reaction medium in the biological catalytic system. Compared with traditional enzyme catalysis in water, non-aqueous phase catalysis can effectively inhibit adverse side reactions, so that the reaction tends to proceed in the direction of synthesis. In addition, natural enzymes often have low biological catalytic activity in the catalysis of unnatural substrates, which seriously limits the industrial application of enzymes. With the continuous breakthrough and development of biological catalysis field, structure-based protein engineering can effectively modify enzyme molecules to obtain high-efficiency biological catalysts.

[0006] According to the mechanism of the reaction, the amidation reaction of p-hydroxycinnamic acid and its derivatives with o-aminobenzoic acid can realize the synthesis of corresponding dihydroheteroclausine D. There are many kinds of enzymes that can realize this catalytic process, including lipase, acyltransferase, amide synthetase, carboxylate reductase, penicillin acylase and aminoacylase, etc. However, there is no report on the enzyme-catalyzed research and production of dihydroheteroclausine D. SUMMARY

[0007] The purpose of the present application is to provide a lipase mutant and its application and method in the biological catalytic preparation of dihydroheteroclausine.

[0008] In order to achieve the above-mentioned purpose of the application, the technical solutions of the present application are as follows:

[0009] On the one hand, the present application provides a lipase mutant, and the amino acid sequence of the lipase mutant is shown in SEQ ID NO: 2.

[0010] Specifically, the lipase mutant is a derivative protein formed by replacing the proline at position 286 of the amino acid sequence shown in SEQ ID NO: 1 with alanine.

[0011] SEQ ID NO: 1:

[0012] MRRRPGLDTASRCFRGIRYATAERFQPAELLGFEGLDGDRGRGPVSPQDPSRLDMIMGPAAQLAQSEHCQVLSVFTPSLDGRRPVMVWLHGGAFVSGGGELPWYDAARLSAEQDVVVVTVTYRLGAFGFLQLADTAGPSPGTSDQIAALQWVHRHIGQFGGDPDNVTLFGHSAGGASIEAILQWGHASLVRRAILQSGNARTHRRTREEADKVAQMFVGLTGTDPRLLAEDEILPIQRELSRGRHFAFDWWPTSPDVPAEFAVDLMAGWTRDDGLPFLMLADQARPGPDTLSSYSDRIGPANAMFSVGSHAAAQAAVAAGRRAWLYRFDWEAPASHLGAPHCIELPFLLGEPEAWTAAPMLAGASWHEIDSLGAGLRAAWASFARGDGPGDAWRACSDADQPLNILPMRDKGRNP

[0013] SEQ ID NO: 2:

[0014] MRRRPGLDTASRCFRGIRYATAERFQPAELLGFEGLDGDRGRGPVSPQDPSRLDMIMGPAAQLAQSEHCQVLSVFTPSLDGRRPVMVWLHGGAFVSGGGELPWYDAARLSAEQDVVVVTVTYRLGAFGFLQLADTAGPSPGTSDQIAALQWVHRHIGQFGGDPDNVTLFGHSAGGASIEAILQWGHASLVRRAILQSGNARTHRRTREEADKVAQMFVGLTGTDPRLLAEDEILPIQRELSRGRHFAFDWWPTSPDVPAEFAVDLMAGWTRDDGLPFLMLADQARPGPDTLSSYSDRIGPANAMFSVGSHAAAQAAVAAGRRAWLYRFDWEAPASHLGAPHCIELPFLLGEPEAWTAAAMLAGASWHEIDSLGAGLRAAWASFARGDGPGDAWRACSDADQPLNILPMRDKGRNP

[0015] In yet another aspect, the present application provides a gene encoding the above-mentioned lipase mutant, the nucleotide sequence of the gene is shown as SEQ ID NO: 3.

[0016] SEQ ID NO:3:

[0017]

[0018] In another aspect, the present application provides an expression vector comprising the gene described above.

[0019] Specifically, the expression vector includes but is not limited to a recombinant expression vector and a recombinant expression transformant.

[0020] Specifically, the present application provides a host cell comprising the recombinant expression vector described above.

[0021] In another aspect, the present application provides a method for the enzymatic synthesis of dihydrohuperzine D in a non-aqueous phase medium, using an organic solvent as the reaction medium, adding methyl p-hydroxybenzoate and o-aminobenzoic acid, and using the recombinant lipase mutant represented by SEQ ID NO: 2 described above as a catalyst to synthesize dihydrohuperzine D.

[0022] Specifically, the organic solvent is selected from one or more of n-pentane, n-hexane, cyclohexane, isooctane, dichloromethane, toluene, o-xylene, diethyl ether, dibenzyl ether, and methyl tert-butyl ether.

[0023] Further, the organic solvent is methyl tert-butyl ether.

[0024] Specifically, the reaction concentration of methyl p-hydroxybenzoate is 1-50 mmol / L.

[0025] Further, the reaction concentration of methyl p-hydroxybenzoate is 10-30 mmol / L.

[0026] Still further, the reaction concentration of methyl p-hydroxybenzoate is 20 mmol / L.

[0027] Specifically, the reaction concentration of o-aminobenzoic acid is 1-80 mmol / L.

[0028] Further, the reaction concentration of o-aminobenzoic acid is 50-70 mmol / L.

[0029] Still further, the reaction concentration of o-aminobenzoic acid is 60 mmol / L.

[0030] Specifically, the molar ratio of the amount of o-aminobenzoic acid and methyl p-hydroxybenzoate is 1-5:1.

[0031] Further, the molar ratio of the amount of o-aminobenzoic acid and methyl p-hydroxybenzoate is 3:1.

[0032] Specifically, the concentration of the recombinant lipase mutant is 12-24 g / L.

[0033] According to some embodiments of the present application, the concentration of the recombinant lipase mutant is 12 g / L, 16 g / L, 20 g / L, 24 g / L.

[0034] Further, the concentration of the recombinant lipase mutant is 16-24 g / L.

[0035] Still further, the concentration of the recombinant lipase mutant is 16 g / L.

[0036] Specifically, the recombinant lipase mutant is from a recombinant Escherichia coli lipase expression strain.

[0037] Specifically, the reaction condition is constant temperature, and the reaction temperature is 25-40℃.

[0038] According to some embodiments of the present application, the reaction temperature is 25℃, 30℃, 37℃, 40℃.

[0039] Further, the reaction temperature is 37℃.

[0040] Specifically, the oscillation condition is 100-300 rpm.

[0041] According to some embodiments of the present application, the oscillation condition is 100 rpm, 175 rpm, 200 rpm, 250 rpm, 300 rpm.

[0042] Further, the oscillation condition is 250 rpm.

[0043] Specifically, the reaction time is 6-24 h.

[0044] According to some embodiments of the present application, the reaction time is 6 h, 8 h, 10 h, 12 h, 24 h.

[0045] Further, the reaction time is 12 h.

[0046] According to some embodiments of the present application, the technical route for synthesizing dihydrohuperzine D is as follows:

[0047]

[0048] Specifically, after the reaction is completed, high performance liquid chromatography is used for analysis.

[0049] Further, the detection condition of the high performance liquid chromatography is as follows:

[0050] (1) Column: XDB-C18;

[0051] (2) Mobile phase: acetonitrile: water (1‰ trifluoroacetic acid) = 45:55;

[0052] (3) Column temperature: 30℃;

[0053] (4) Detection wavelength: 254nm;

[0054] (5) Injection volume: 20μL.

[0055] In another aspect, the present application provides dihydro avenacoside D prepared by the above method.

[0056] Specifically, the dihydro avenacoside D has an amide bond structure of methyl p-hydroxybenzoate and anthranilic acid.

[0057] The present application has the following advantages:

[0058] The present application relates to engineering design of a wild-type recombinant lipase from Rhizorhabdus wittichii, using molecular docking and "alanine scanning" to modify the active center of the wild-type recombinant lipase, replacing the proline at position 286 of the amino acid sequence shown in SEQ ID NO: 1 with alanine to form a derivative protein, the new amino acid sequence of which is shown in SEQ ID NO: 2, to obtain a recombinant lipase mutant P286A, which realizes the biosynthesis of dihydro avenacoside D for the first time, and compared with the traditional chemical synthesis of dihydro avenacoside D, the present method uses enzyme method for one-step synthesis, without the need for group protection and deprotection steps, and the production process is green and environmentally friendly, greatly reduces the discharge of industrial wastewater, and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 It is a high performance liquid chromatogram of methyl p-hydroxybenzoate, and the retention time of the substrate peak is about 6.999.

[0060] Figure 2 It is a high performance liquid chromatogram of anthranilic acid, and the retention time of the substrate peak is about 3.976.

[0061] Figure 3 It is a high performance liquid chromatogram of dihydro avenacoside D, and the retention time of the product peak is about 9.420.

[0062] Figure 4 It is the screening result of the recombinant lipase mutant.

[0063] Figure 5 It is a reaction result graph of anthranilic acid and methyl p-hydroxybenzoate in different proportions.

[0064] Figure 6 It is a reaction result graph of different amounts of engineered E. coli of recombinant lipase.

[0065] Figure 7 Reaction results chart for different reaction temperatures.

[0066] Figure 8 Reaction results chart for different oscillation conditions. DETAILED DESCRIPTION

[0067] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the following specific embodiments are further described, but the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application. In the following embodiments, if not otherwise specified, the operation method used is a conventional operation method, the equipment used is a conventional equipment, and the equipment materials used in each embodiment are the same.

[0068] Example 1 Preparation of reaction catalyst

[0069] The recombinant E. coli lipase expression strain (host strain BL21 DE3, Qianke Biology) was activated, induced expression, and freeze-dried bacteria powder and crude enzyme solution were prepared.

[0070] Strain activation: the recombinant E. coli lipase expression strain was inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C, 200 rpm for 12 hours. Then, 0.5 mL of the culture was inoculated into 50 mL of LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C, 200 rpm for about 2.5 hours.

[0071] Induction expression: when the OD600 value was 0.6-0.8, the inducer IPTG (final concentration 0.1 mM) was added, and the culture was incubated at 20°C, 200 rpm for 18-22 hours. After the end, the bacterial solution was transferred to a centrifuge tube, and after balancing, the bacteria were collected by centrifugation (8000 rpm, 10 min), and the supernatant was discarded. After resuspension and washing twice with physiological saline, the supernatant was discarded.

[0072] Preparation of freeze-dried bacteria powder: after being placed in a -40°C refrigerator for at least 6 hours, the bacteria were prepared into freeze-dried bacteria powder by using a freeze dryer.

[0073] Preparation of crude enzyme solution: according to the mass of wet bacteria, add phosphate buffer (100 mM, pH 7.0) in proportion to resuspend, put into ice box for crushing. The program of ultrasonic cell crusher is set to work for 3 seconds interval 5 seconds, crushing for 15 minutes, and the power is set to 250-300 W. After crushing, centrifuge at 8000 rpm for 15 minutes, add 4 mL of 75% glycerol to each 8 mL of supernatant, mix evenly, then distribute to EP tubes, and store in -40°C refrigerator for standby, and the precipitate is resuspended with equal volume of sodium phosphate buffer for standby.

[0074] Example 2 Screening of catalysts

[0075] The reaction was carried out at 37°C in a 150 rpm shaker, and a 1 mL buffer screening system was established. A 2 mL EP tube was used as the reaction container, 10 mmol / L methyl p-hydroxybenzoate, 50 mmol / L o-aminobenzoic acid, 10% dimethyl sulfoxide as reaction cosolvent and 1 mL crude enzyme solution were added to the container, and after thorough shaking and vortexing, the reaction was carried out in a shaker for 12 hours. After the reaction was completed, the sample was placed in a 90°C water bath for 10 minutes, two times the amount of dimethyl sulfoxide was added to the sample, and after the end of the reaction, the sample was centrifuged (8000 rpm, 10 min) to obtain the supernatant, which was then analyzed by high performance liquid chromatography.

[0076] The detection conditions of high performance liquid chromatography are as follows:

[0077] (1) Chromatographic column: XDB-C18;

[0078] (2) Mobile phase: acetonitrile: water (1‰ trifluoroacetic acid) = 45:55;

[0079] (3) Column temperature: 30°C;

[0080] (4) Detection wavelength: 254 nm;

[0081] (5) Injection volume: 20 μL.

[0082] A wild-type recombinant lipase was screened from 69 lipase enzyme libraries. The wild-type lipase (amino acid sequence as shown in SEQ ID NO: 1) had a conversion rate of 35% in the process of catalyzing methyl p-hydroxybenzoate and o-aminobenzoic acid after 12 hours of reaction, and had the preliminary biological activity of synthesizing dihydrohuperzine D. The high performance liquid chromatography analysis chart of methyl p-hydroxybenzoate is shown in Figure 1 , the high performance liquid chromatography analysis chart of o-aminobenzoic acid is shown in Figure 2 , and the high performance liquid chromatography analysis chart of dihydrohuperzine D is shown in Figure 3 .

[0083] Example 3 Screening of organic solvents

[0084] The raw materials of the reaction, methyl p-hydroxybenzoate and o-aminobenzoic acid, are difficult to dissolve in water, and the lipase-mediated enzymatic reaction in aqueous phase can cause product decomposition, while using organic solvents as reaction medium can effectively inhibit adverse side reactions, making the reaction tend to proceed in the direction of synthesis. Considering that polar solvents can cause enzyme denaturation and inactivation, in order to find a suitable organic solvent, ten non-polar solvents, n-pentane, n-hexane, cyclohexane, isooctane, dichloromethane, toluene, o-xylene, diethyl ether, dibenzyl ether and methyl tert-butyl ether, were selected as reaction media, and a screening system was established.

[0085] In a 1 mL reaction screening system, 2 mL EP tubes were used as reaction containers, 10 mmol / L methyl p-hydroxybenzoate and 50 mmol / L o-aminobenzoic acid were added to the tubes, 1 mL of the reaction system was supplemented with organic solvents, and 24 g / L wild-type recombinant lipase engineered E. coli was used as the reaction catalyst. The reaction temperature and time were 37°C and 12 hours, respectively. After the reaction was completed, the results were analyzed by high performance liquid chromatography (the conditions of high performance liquid chromatograph were the same as those in Example 2). The results showed that the wild-type recombinant lipase only exhibited catalytic activity in the reaction medium of methyl tert-butyl ether, and the conversion rate reached 43% after 12 hours of reaction, while it did not exhibit obvious catalytic activity in other organic solvents. Therefore, methyl tert-butyl ether was selected to establish a non-aqueous phase catalytic system to synthesize dihydrohuperzine D.

[0086] Example 4 Engineering design of wild-type recombinant lipase

[0087] In order to obtain a biocatalyst that can be applied to industrial production, various protein engineering methods including molecular docking and alanine scanning were used to modify the wild-type recombinant lipase. First, the protein model of the wild-type recombinant lipase was established using an online website (https: / / swissmodel.expasy.org / ), and the model structures of methyl p-hydroxybenzoate and o-aminobenzoic acid were obtained from the organic small molecule bioactivity database (https: / / pubchem.ncbi.nlm.nih.gov / ). Then, molecular docking was performed using the computational software AutoDock 4.2.6, and the results were analyzed by visualization software to determine the 10 amino acid residues that constitute the active center of the wild-type recombinant lipase. Considering that the non-natural substrate may face unfavorable steric hindrance when binding to the active center of the wild-type recombinant lipase, which can affect the catalytic efficiency of the substrate, the 10 amino acid residues in the active center were mutated to alanine by the method of "alanine scanning".

[0088] The plasmid of the wild-type recombinant lipase was used as the parent, and primers were designed for the mutation sites as shown in Table 1.

[0089] Table 1

[0090] Primer Sequence (5'-3') SEQ ID NO: D54AF cgccggctgaaaggcttccgcggtcgca SEQ ID NO:4 D54AR tgcgaccgcggaagcctttcagccggcg SEQ ID NO:5 M55A F ccagatgatcttccaaacgaaaggccagaccattc SEQ ID NO:6 M55AR gaatggtctggcctttcgtttggaagatcatctgg SEQ ID NO:7 I56A F gcccaacgacgctgaaacggatcgccatctgc SEQ ID NO:8 I56A R gcagatggcgatccgtttcagcgtcgttgggc SEQ ID NO:9 M57A F gtatccaaacctgggctcctccgcatgga SEQ ID NO:10 M57A R tccatgcggaggagcccaggtttggatac SEQ ID NO:11 G58AF agcagttcgccgcccttaataattgcaacattaaagcccggacc SEQ ID NO:12 G58AR ggtccgggctttaatgttgcaattattaagggcggcgaactgct SEQ ID NO:13 P286AF tcatcaatgcgcgtaaacggccaaataccgcctgcgg SEQ ID NO:14 P286AR ccgcaggcggtatttggccgtttacgcgcattgatga SEQ ID NO:15 S335AF aatgcgtgcatccgataacggaacaaattcgccatccaca SEQ ID NO:16 S335AR tgtggatggcgaatttgttccgttatcggatgcacgcatt SEQ ID NO:17 L337AF ccccactgaaaatttttaaccgtcggatcaatcacattacgcgg SEQ ID NO:18 L337AR ccgcgtaatgtgattgatccgacggttaaaaattttcagtgggg SEQ ID NO:19 M360A F gtatgcggaccaggcttaccatcaccaataacggtatcatca SEQ ID NO:20 M360A R tgatgataccgttattggtgatggtaagcctggtccgcatac SEQ ID NO: 21 L361A F taaaatggcgctatcataaccgcgatcgcctgcttcctg SEQ ID NO: 22 L361A R caggaagcaggcgatcgcggttatgatagcgccatttta SEQ ID NO: 23

[0091] The PCR reaction system is shown in Table 2 below:

[0092] Table 2.

[0093]

[0094]

[0095] PCR reaction conditions: 95°C pre-denaturation for 3 min; 98°C denaturation for 5 s, 58°C annealing for 15 s, 72°C extension for 5 min, this stage is cycled for 30 times; final 72°C terminal extension for 5 min; 4°C preservation.

[0096] After the PCR amplification is completed and verified as positive by agarose gel electrophoresis, 0.5 μL of DpnI enzyme is added to specifically cut off the methylated template DNA, and placed in a 37°C metal bath for 3 h. Subsequently, transformation, bacteria picking, and sequencing can be performed, and the corresponding recombinant lipase mutant is prepared by the method described in Example 1.

[0097] A 1 mL reaction system is set up for screening the mutant, a 2 mL EP tube is used as the reaction container, 10 mmol / L methyl p-hydroxybenzoate and 50 mmol / L o-aminobenzoic acid are added to the tube, 1 mL of the reaction system is supplemented with methyl tert-butyl ether, and 24 g / L (dry weight) of engineered E. coli recombinant lipase is used as the reaction catalyst, and the reaction temperature and time are 37°C and 12 hours, respectively.

[0098] The reaction results are shown in Table 3. Figure 4 After 12 hours of reaction, the reaction conversion rate of the recombinant lipase mutant P286A is increased from 43% to 68% compared with the wild type, indicating that modification of the active pocket can effectively improve the binding efficiency of the substrate and the enzyme.

[0099] Determination of the optimal synthesis conditions

[0100] Since o-aminobenzoic acid is difficult to dissolve, the reaction concentration is selected as 60 mmol / L.

[0101] In the experiment of different molar ratios of o-aminobenzoic acid and methyl p-hydroxybenzoate input (Table 4), Figure 5 the reaction conversion rate reaches the highest of 72% when the molar ratio of input is 3:1, and therefore the optimal reaction concentration of methyl p-hydroxybenzoate is 20 mmol / L.

[0102] The engineered E. coli addition amount of the optimized recombinant lipase mutant P286A is selected as 8 g / L, 12 g / L, 16 g / L, 20 g / L and 24 g / L in the experiment as a single variable of the reaction, and the experimental results are shown in Table 2. Figure 6 As shown in Table 2, when the engineered E. coli addition amount of the recombinant lipase is increased from 8 g / L to 16 g / L, the reaction conversion rate is increased from 54% to 72%, and when the engineered E. coli addition amount of the recombinant lipase is further increased, the reaction conversion rate is not obviously increased, so 16 g / L is selected as the optimal reaction condition.

[0103] The reaction temperature is optimized, and 25℃, 30℃, 37℃, 40℃, 45℃ and 50℃ are selected as the reaction temperature in the experiment, and the results are shown in Table 3. Figure 7 As shown in Table 3, the reaction conversion rate is the highest at 37℃ and reaches 72%, so 37℃ is selected as the optimal condition.

[0104] The shaking condition is optimized, and 100 rpm, 175 rpm, 200 rpm, 250 rpm and 300 rpm are selected as the shaking condition in the experiment, and the results are shown in Table 4. Figure 8 As shown in Table 4, the reaction conversion rate is the highest at 250 rpm and reaches 72%.

[0105] The above only describes the preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A lipase mutant, characterized in that, The amino acid sequence of the lipase mutant is shown as SEQ ID NO:

2.

2. A gene encoding the lipase mutant of claim 1, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO:

3.

3. An expression vector, characterized by, The expression vector comprises the gene of claim 2.

4. A method for the enzymatic synthesis of dihydrolupanine D in a non-aqueous phase medium, characterized in that, Dihydro-oat alkaloid D is synthesized by using the lipase mutant of claim 1 as catalyst, adding methyl p-hydroxybenzoate and o-aminobenzoic acid, and using an organic solvent as reaction medium.

5. The method of claim 4, wherein, The organic solvent is selected from one or more of n-pentane, n-hexane, cyclohexane, isooctane, dichloromethane, toluene, o-xylene, diethyl ether, dibenzyl ether, and methyl tert-butyl ether.

6. The method of claim 5, wherein, The organic solvent is methyl tert-butyl ether.

7. The method of claim 4, wherein, The reaction concentration of the methyl p-hydroxybenzoate is 1-50 mmol / L; and the reaction concentration of the o-aminobenzoic acid is 1-80 mmol / L.

8. The method of claim 7, wherein, The reaction concentration of the methyl p-hydroxybenzoate is 20 mmol / L; and the reaction concentration of the o-aminobenzoic acid is 60 mmol / L.

9. The method of claim 4, wherein, The molar ratio of the input amount of the o-aminobenzoic acid to the methyl p-hydroxybenzoate is 1-5:

1.

10. The method of claim 9, wherein, The molar ratio of the input amount of the o-aminobenzoic acid to the methyl p-hydroxybenzoate is 3:

1.

11. The method of claim 4, wherein, The concentration of the lipase mutant is 12-24 g / L.

12. The method of claim 11, wherein, The concentration of the lipase mutant is 16-24 g / L.

13. The method of claim 12, wherein, The concentration of the lipase mutant is 16 g / L.

14. The method of claim 4, wherein, The reaction condition is constant temperature, the reaction temperature is 25-40 ℃, the oscillation condition is 100-300 rpm, and the reaction time is 6-24 h.

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